An efficient method for the site-specific 99mTc labeling of nanobody

Recently, there has been a lot of interest by using nanobodies (heavy chain-only antibodies produced naturally from the Camelidae) as targeting molecules for molecular imaging, especially for the nuclear medicine imaging. A radiolabeled method that generates a homogeneous product is of utmost importance in radiotracer development for the nuclear medicine imaging. The conventional method for the radiolabeling of nanobodies is non-specifically, which conjugates the radioisotope chelating group to the side chain ɛ-amine group of lysine or sulfhydryl of cysteine of nanobodies, with a shortcoming of produce of the heterogeneous radiotracer. Here we describe a method for the site-specific radioisotope 99mTc labeling of nanobodies by transpeptidase Sortase A. The radiolabeling process includes two steps: first step, NH2-GGGGK(HYNIC)-COOH peptide (GGGGK = NH2-Gly-Gly-Gly-Gly-Lys-COOH, HYNIC = 6-hydrazinonicotinyl) was labeled with 99mTc to obtain GGGGK-HYNIC-99mTc; second step, Sortase A catalyzes the formation of a new peptide bond between the peptide motif LPETG (NH2-Leu-Pro-Glu-Thr-Gly-COOH) expressed C-terminally on the nanobody and the N-terminal of GGGGK-HYNIC-99mTc. After a simple purification process, homogeneous single-conjugated and stable 99mTc-labeled nanobodies were obtained in >50% yield. This approach demonstrates that the Sortase A-mediated conjugation is a valuable strategy for the development of site-specifically 99mTc-labeled nanobodies.


INTRODUCTION
Recently, a new class of variable region of the heavychain-only antibodies (V H H) derived from Camelidae, referred to as nanobody (Nb) (Hamers-Casterman et al. 1993), has gained a growing interest in the field of molecular imaging, given their peculiar features and high versatility (Yang and Shah 2020). The main advantages of Nb as molecular probes are as follows: (1) Compared with the monoclonal antibodies (mAbs,~1 50 kDa), antigen fragment (Fab, ~50 kDa) and single chain Fv (sc-Fv, ~25 kDa), Nb has the smallest molecular weight (12-15 kDa) ( Fig. 1) (Oliveira et al. 2013). Due to their small size and the absence of Fc fragment, Nb is rapidly eliminated from the circulation, and can be cleared quickly through the kidney, which results in a significantly reduced background and increased signal-to-noise ratio as early as 1 h after tracer injection ); (2) Compared with peptides, Nb has high affinity and specificity. The antigen binding affinity of Nb is more than 10-100 times to peptides, which is close to mAbs (Hassanzadeh-Ghassabeh et al. 2013); (3) The immunogenicity and toxicity of Nb are very low, and they are not as prone to adhesion as sc-Fv (Keyaerts et al. 2016); (4) Nb has good tissue penetration and can be fully combined to targeted tissues; (5) By using modern genetically engineered antibody technology, high yield Nb can be obtained (McMahon et al. 2018), whose structure can also be easily modified, making it an ideal targeting molecule candidate for nuclear medicine imaging agents.
The previously reported methods of Nb radiolabeling are usually accomplished by using the side chain primary amine of lysine residues or sulfhydryl of cysteine residues of Nb (Lv et al. 2020), but these methods have some limitations. Nb usually has multiple solvent-exposed lysine, making it difficult to control where and how many radioisotopes are labeled. In addition, the presence of lysine residues at or near the target antigen-binding site can lead to reduced Nb activity after conjugation . In order to avoid the heterogeneity of the tracer, some studies have introduced the unpaired cysteine at the C-terminal of Nb for site-specific labeling (Feng et al. 2020). However, this strategy requires the reducing agent to liberate the introduced cysteine residue. These reducing agents must be carefully titrated to prevent the breakdown of disulfide bonds within Nb, which are essential for stability and may lead to unnecessary reduction by-products. Other methods under investigation for designing site-specific labeling of Nb are alkyne-azide click reactions, which involve the insertion of unnatural amino acids into the nanobody structure (Agarwal and Bertozzi 2015). In addition, 99m Tc-tricarbonyl reacts site-specifically with a genetically inserted C-terminal hexahistidine tag (His 6 ) of nanobody for 99m Tc labeling (Xing et al. 2019).
Sortase A (SrtA), a transpeptidase, is derived from Staphylococcus aureus that has been extensively used for protein engineering and antibody modification Popp et al. 2007). SrtA recognizes substrate proteins bearing a short motif (LPXTG) of Cterminal and cleaves the peptide between threonine and glycine forming a new bond with the nucleophiles containing N-terminal oligo-glycine motif (Mazmanian et al. 1999). Several studies have reported the use of SrtA for the site-specific labeling of Nb (Massa et al. 2016;Rashidian et al. 2016). For example, Massa et al. demonstrated SrtA-mediated the site-specific indium-111 and gallium-68 labeling of human epidermal growth factor receptor 2 (HER2)-targeting nanobody (Massa et al. 2016). Since nearly 85% of diagnostic radiotracers currently available in clinical nuclear medicine are 99m Tc-compounds due to the ideal nuclear properties of 99m Tc, as well as their widespread availability using commercially available 99m Tcgenerators (Pietzsch et al. 2013). Here, we describe a generic method for SrtA-mediated site-specific 99m Tc labeling of Nb, while using the programmed death ligand-1 (PD-L1)-targeting nanobody (MY1523). First step, NH 2 -GGGGK(HYNIC)-COOH peptide was labeled with 99m Tc using TPPTS and tricine as co-ligands to obtain trinary 99m Tc-radiolabed complex of ( 99m Tc-(HYNIC-peptide) (TPPTS)(tricine)) (termed as GGGGK-HYNIC-99m Tc). This trinary 99m Tc-radiolabed complex have been reported to have good stability (Jia et al. 2006). Second step, the SrtA catalyzes the formation of a new peptide bond between the peptide motif LPETG expressed C-terminally on the MY1523 and the Nterminal of GGGGK-HYNIC-99m Tc (Fig. 2). This enzymemediated ligation is a more elegant method which avoids Nb to contact violent labeling conditions. We expect this labeling protocol to be resulted in a homogeneous, site-specifically single-conjugated, and stable 99m Tc-labeled nanobody.
[CRITICAL STEP] It is highly recommended to maintain the duration of the reaction between 25-30 min. Prolonging the reaction time will reduce the labeling efficiency.
Step 14-20 Preparation and determination of the radiochemical yield of GGGGK-HYNIC-99m Tc take approximately 1 h.
Step 21-29 Preparation and determination of the radiochemical yield of 99m Tc-MY1523 take approximately 1 h.
Step 33-40 Determination of the radiochemical purity of 99m Tc-MY1523 takes approximately 1 h.
Step 41-44 Assessment of the in vitro stability of 99m Tc-MY1523 takes approximately 24 h.
Step 45-48 Assessment of the in vivo stability of 99m Tc-MY1523 takes approximately 6-7 h.

[? TROUBLESHOOTING]
Step 22 When CaCl 2 water solution is added, white fluffy precipitate was detected in the mixed solution. This is Ca 2+ precipitate (Ga 3 (PO 4 ) 2 ). We can centrifuge the mixture, take the supernatant and continue the labeling reaction.
Step 29 If the labeling efficiency is very low, it may be that, (1) insufficient GGGGK-HYNIC-99m Tc is added to the reaction, (2) pH of the reaction solution is not compatible with SrtA activity, or (3) SrtA is inactive. We can try that, (1) increase the amount of GGGGK-HYNIC-99m Tc, (2) ensure the pH of the reaction solution is 7-8, or (3) use new SrtA.
First, determine whether the radiochemical yield was within the expected range (>50%). One possibility is that the nanobody was stuck on the Superdex-75 TM column. In this case, using 0.1% Tween-20-PBS (pH = 7.2-7.4) as mobile phase can help flush out the radiolabeled nanobody.

ANTICIPATED RESULTS
Figures 3 and 4 present typical representative data obtained using the method described here. SrtA mediated the site-specific radionuclide 99m Tc labeling of nanobody.
The radiochemical yield (RCY) of GGGGK-HYNIC-99m Tc was determined by RP-HPLC. The representative HPLC chromatogram of GGGGK-HYNIC-99m Tc was shown in Fig. 3A. The RCY of GGGGK-HYNIC-99m Tc was >95%. The RCY of 99m Tc-MY1523 was determined by ITLC. The representative ITLC chromatogram was shown in Fig. 3B. As results, 50% RCY was generally obtained after the two steps in total. After purification, the radiochemical purity (RCP) of the final product was determined by ITLC. The representative ITLC chromatogram of 99m Tc-MY1523 was shown in Fig. 3C. The RCP of end-product was >95%. The specific activity of 99m Tc-MY1523 was >11.0 MBq/nmol. The RCP of 99m Tc-MY1523 was also determined by HPSEC. Fig. 4 shows a typical representative HPSEC chromatogram. The RCP of 99m Tc-MY1523 was >95%. The retention time of 99m Tc-MY1523 was at 16.6 min, which was a little earlier than that of cold MY1523 (17.3 min for MY1523). As shown in Fig. 5A, 99m Tc-MY1523 was stable in mouse serum at room temperature for 24 h. The RCP of urine sample collected at 6 h p.i. was >95% (Fig. 5B), indicating that 99m Tc-MY1523 has good in vivo stability.    • Electric-heated thermostatic water bath (Shanghai Senxin Experimental Instrument Co, Ltd, cat. no. DK-S12)

MATERIALS AND EQUIPENT
• Dry bath incubator (Fisher Scientific, • ITLC-SG chromatograpy paper (10 cm long and 1.5 cm wide, Agilent Technologies, cat. no. SGI0001) • pH paper ( • 100 mmol/L NH 4 OAc buffer (pH = 7.0). Use ammonium hydroxide to adjust the pH of 100 mmol/L NH 4 OAc water solution to 7. Filter the buffet and store it at 4 °C for up to three months.
• 20% Piperidine-DMF (v/v). Mix 20 mL of piperidine with 80 mL of DMF. The Fmoc deprotection solution can be stored at RT for one month.
• 0.1% Tween-20-PBS (v/v). Mix 1 mL of Tween-20 with 1 L of PBS. Filter the solution and store it at 4 °C for up to three months.